RU2084152C1 - Strain of bacterium bacillus subtilis used for plant protection from phytopathogenic bacteria - Google Patents

Abstract

FIELD: agriculture microbiology. SUBSTANCE: strain of bacterium Bacillus subtilis M-22 is isolated from the environment, deposited in All-Russian Collection of Institute of Plant Protection at N M-22 and shows antagonistic activity with respect to the following microorganisms: Clavibacter michiganense subsp. michiganense, Xanthomonas campestris pv. vesicatora, Pseudomonas corrugata, Pseudomonas syringae pv. tomato, Erwinia carotovora subsp. carotovora, Alternaria solani, Verticillium dachliae, Fusarium graminearum, Ascohyta fabae, Colletotrichum lagenarium, Rhizoctonia solani, Bipolaris sorokiniana, Sclerotinia sclerotiorum, Sertoria nodorum. EFFECT: enhanced effectiveness. 4 tbl

Description

 The invention relates to agricultural mycobiology and is a bacterial strain that is an antagonist to the poison of phytopathogenic bacteria. The strain B. subtilis was deposited in the collection VIZR under N 22. Identification of the strain was carried out according to the “Brief identifier of bacteria Bergi", "Mir", M. 1980

 Chemical pesticides are low in protection against bacterial diseases of crops and significant toxicity to warm-blooded animals and humans, therefore their use is limited.

 Currently, microbiological preparations based on phytolavin and phytobacteriomycin antagonist strains are used to a limited extent against bacteriosis.

 The main disadvantage of these drugs is low biological activity and a narrow spectrum of diseases. ("List of chemical and biological agents for controlling pests, diseases and weeds, and plant growth regulators approved for use in agriculture for 1986 1990." M. 1987, pp. 95-96).

 Closest to the proposed is a strain of Bacillus subtilis IMP-215, which is a producer of bactophite (baciphyte) for protection against powdery mildew of cucumbers, cotton verticillosis, root rot of vegetable and grain crops ("Problems of the creation and use of microorganic plant protection products", M. 1989, pp. 253, 257-295_ The disadvantage of this strain is its lack of effectiveness against phytopathogenic bacteria.

 The strain Bacillus subtilis M-22-VIZR has a high antagonistic activity against a wide range of pathogens of plant bacterioses and is a producer of the biological product gamair. The strain is isolated from the environment in Kiev (Ukraine).

 Cultural and morphological characters.

 The strain B. subtilis M-22-VIZR has the following morphological and cultural characteristics: cells are spore-forming bacilli, rarely located in chains, the location of the endospore is central. Cell sizes are 0.8-0.9 μm x 1.7-2 μm. The sizes of endospores are 0.7-0.8 microns x 1.0-1.4 microns. Gram stains positively.

To study the cultural and morphological properties of the strain, the culture was grown at 26 ± 2 ^o C for 2-4 days on solid nutrient media of the following composition:
Meat-peptone agar (MPA):
meat-peptone broth 800 ml
tap water 200 ml
glucose 10 g
agar 20 g
sterilization at 0.5 atm 20 min.

Wort agar
200 ml wort
water 800 ml
agar 20 g
sterilization at 0.8 atm 20 min.

Corn and molasses agar:
corn extract 30 g
molasses 15 g
agar 20 g
water 1 l
sterilization at 0.8 atm 20 min.

 pH of the medium before sterilization of 0.8-7.2.

 On agar media on the 1st day, the colonies are small, dull brilliant. On 2-3 days, the growth of the colony on MPA with glucose and corn and molasses agar is dense, irregular in shape, the edge is lobed, the center is folded, dull shiny, the periphery is dull. On glucose-free MPA and wort agar, the colonies are whitish, strongly growing, more emanating, the center is slightly elevated. With the growth of lawn in the early stages of growth, the culture is whitish, leathery. Then the surface becomes tuberous-folded, beige in color.

Physiological characteristics: it has catalase activity, hydrolyzes starch, does not grow under anaerobic conditions, does not have lecithinase activity, the maximum growth temperature is 48 ± 2 ^o C, the minimum is -10-12 ^o C. It grows on artificial nutrient media: MPB, MPA, SPA, multicomponent media containing rapeseed meal hydrolyzate, BVK yeast and corn flour in various proportions. It utilizes glucose, mannitol, starch. It is well absorbed by amino acids, proteins and their hydrolysates, yeast hydrolysates, etc.

The biosynthesis method:
A. Sowing medium:
corn extract 3.0
molasses 1.5%
pH 6.8-7.2
B. Fermentation medium is similar to inoculation.

Cultivation was carried out in deep conditions at a temperature of 26 ± 2 ^o C for 3 days. The inoculum was grown for 24 hours. The culture fluid was freeze-dried.

The method of storage of the strain: in test tubes on the jambs of MPA at a temperature of + 4-6 ^o C or in a lyophilized state in ampoules. The frequency of reseeding 2 times a year, re-laying in ampoules 1 time in 5 years.

Antagonistic properties: inhibit the growth of phytopathogenic bacteria and fungi. To determine the antagonistic activity of the strain was grown on corn-molasses medium for 3 days at t 26 ± 2 ^o C. The activity of the culture fluid, ethanol extract of biomass and the filtrate of the culture fluid was determined by the method of holes. Lysis zones of test cultures sown with a lawn on MPA and Chapek's medium were determined after 1-3 days on bacteria and 3-7 days on fungi. (table 1)
Example 1. The strain B.subtilis M-22-VIZR was grown on a medium containing corn extract 3% molasses 1.5% of the rest tap water, pH before sterilization of 7.0 for 3 days at t 28 ± 2 ^o C. The culture fluid was freeze dried. A yield of dry biomass of 40 g / l with a titer of 3 10 ¹⁰ spores / g was obtained. The obtained biological product (gamair) based on the proposed strain was tested on tomato seeds, artificially infected with the causative agent of soft rot of vegetables, under the provocative conditions of a wet chamber. For this, Aurora seeds were soaked in a suspension of Erwinia carotovora subsp, carotovora with a titer of 10 ⁹ cells / ml for 2 hours. Then, the seeds were treated with a suspension of a sample of a biological product at a concentration of 0.05% for 30 minutes. The control seeds were soaked in water for 2 hours. Phytolavin (0.05%), bactofin (0.05%) and TMTD chemical preparations were used as reference standards. Seeds were placed in Petri dishes on moistened filters and incubated for 12 days at 25 ^o C. As can be seen in table 2, the treatment led to stimulation of development and complete suppression of decay of seedlings. It was significantly more effective than in variants with a reference standard (bactofite and TMTD). Phytolavin under conditions of a wet chamber exhibited high phytotoxicity; therefore, it was not possible to analyze its bactericidal effect under conditions due to decay of the seedling.

Example 2. The biological effectiveness of the drug based on B.subtilis M-22-VIZR was tested in a growing experiment on tomato plants of the Minuet hybrid. Seedlings in the phase of 5-6 true leaves were sprayed with 0.05% suspensions of biological products, and then infected with a bacterial cancer pathogen by injection of 0.01 ml of a suspension of C. miohiganense subsp. michiganense with a titer of 10 ⁹ cells / ml in the sinus of the leaf. During the growing season, the prevalence of bacterial tomato cancer was observed, given the wilting of plants, the appearance of spots, and the drying of leaves. The data are presented in table 3. As can be seen from the table, treatment with a biologic preparation gamair based on B. subtilus M-22-VIZR halved the incidence of tomato bacteriosis, while spraying with bactophytum and phytolavin reduced this indicator only by 1.2-1.7 times.

.Example 3. A comparative evaluation of the effectiveness of a biological product based on a strain of B. subtilis M-22-VIZR was carried out under the conditions of a shallow experiment in the production greenhouses of JSC Leto (St. Petersburg) on tomato plantings. The use of biological products began with the soaking of seeds in 0.05% suspensions for 30 minutes. Then, 2-fold spraying of plants with biological products in the same concentration in the seedling period and once after planting tomatoes at a constant place of growth was carried out. During the observation period, the prevalence of bacterial cancer according to the formula:

.

where P is the prevalence,
n number of diseased plants, pcs;
N total number of registered plants, pcs.

 Before harvesting, the number of fruits on one plant was estimated (Table 4). The data in the table indicate that treatment with gamair halved the prevalence of bacterial cancer of tomato (Clavibacter michiganense subsp. Michiganense), which led to a significant increase in the formation of fruits.

Claims (1)

 The bacterial strain Bacillus subtilis VIZR M-22, used to protect plants from phytopathogenic bacteria.

Images